1. Field of the Invention
The present invention relates to a method for screening an antioxidant using mutant bacteria and chlorophyllide. More specifically, the present invention relates to a method for easy screening of an antioxidant by monitoring growth profiles of certain mutant bacteria in filter discs or medium blocks with addition of chlorophyllide, and an antioxidant screened by the same method.
2. Description of the Related Art
As is generally known to those skilled in the art, reactive oxygen species (ROS) or oxygen-free radicals have undesirable oxidizing properties in the body, by which such molecular species contribute to damage of cell membranes, DNA and diverse cell structures and is responsible, either directly or indirectly, for pathogenic causes of various diseases such as cancer, arteriosclerosis, diabetes, cerebral apoplexy, myocardial infarction, hepatitis, nephritis, atopic diseases, Parkinson's disease, and the like. Superoxide is a reactive oxygen species and is produced by reduction of molecular oxygen to the superoxide anion (O2−) via acceptance of an additional one electron in the course of the oxygen metabolism process in living organisms. Superoxide is highly reactive to thereby result in irreversible destruction of biomaterials constituting the organisms, such as nucleic acids, proteins, and lipids. In particular, superoxide reduces Fe3+ ions into Fe2+ ions when Fe3+ ions are present, and the thus-reduced Fe2+ ions react with hydrogen peroxide to form hydroxy radicals. The hydroxy radicals thus formed react with DNA molecules in vivo, which consequently is known to bring about inhibition of metabolism and homeostasis of the organisms, such as in vivo mutagenesis, destruction of iron-sulfur (Fe/S) centers of proteins, peroxidation of lipids, and the like (Imlay, J. A., 2003, Ann. Rev. Microbiol. 57: 395-418).
Therefore, a great deal of importance is given to antioxidant materials which are capable of reducing the cytotoxicity of reactive oxygen species including superoxide anion radicals or are capable of directly inactivating the reactive oxygen species, and various methods have been developed for screening desired antioxidants. For example, antioxidants have been assayed and developed by a method using changes in colors or fluorescence of putative candidate materials through the in vitro redox reaction, a method of using DNA chain cleavage or a method of using spin trap agents which are capable of specifically reacting with reactive oxygen species. However, most of the antioxidants which were developed by these methods suffer from limited uses and applications thereof due to low bioavailability arising from substantial in vivo absorption or the potential risk of toxicity to the organisms. Further, because the aforementioned methods involve in vitro examination of an antioxidant activity, in vivo efficiency, safety and antioxidant specificity of the antioxidant candidate must be verified by repeated multiple screening processes.
In spite of excellent biomembrane permeability and bioavailability as well as a high antioxidant activity, fat-soluble antioxidants may cause deleterious effects on cell body since they are mostly localized in diverse intracellular membranes, which makes it difficult to exert the antioxidant action at aqueous target site where reactive oxygen species are produced. Also, it is difficult for the fat-soluble antioxidants to be metabolized into more hydrophilic forms in liver after reacting with ROS or RNS (reactive nitrogen species), which facilitate excretion of the hydrophobic chemicals from the body. On the other hand, the water-soluble antioxidants exhibit smooth external excretion of the oxidized antioxidants, but have low biomembrane permeability which raises various problems associated with cell membrane-protective effects and difficulty of diffusion of the antioxidant into intracellular organelles. Therefore, there is a need for development of an amphiphilic antioxidant which can be easily formulated into a desired antioxidant preparation, by having both water-solubility and fat-solubility in order to comply with desired uses and applications of the antioxidant.
Meanwhile, microorganisms of the genus Rhodobacter which are purple non-sulfur photosynthetic bacteria exhibit advantages such as no evolution of oxygen during the photosynthetic process unlike algal or plant photosynthesis, and capability to grow and proliferate under various culture conditions, e.g. aerobic conditions, anaerobic dark conditions and anaerobic light conditions. In particular, the members of the phototrophic Rhodobacter genus carry out a series of cell membrane-associated electron transfer processes by absorption of long-wavelength light via the photosynthetic apparatus containing bacteriochlorophyll pigments under the anaerobic light conditions, which results in conversion of light energy into chemical energy, and the thus-generated chemical energy is used as energy necessary for a variety of cell metabolic activities such as carbon dioxide fixation. In the photosynthetic bacteria, a multimeric protein, which is one of the enzymes that mediate the metabolic process for synthesis of bacteriochlorophyll and is produced by expression of three genes bchX, bchY and bchZ, is known to have chlorophyllide-reducing activities.
Korean Patent Application Publication No. 2007-59485 A1 discloses a multimeric protein as a superoxide-generating enzyme derived from Rhodobacter sphaeroides. According to this patent, the multimeric protein produces superoxide in vivo and in vitro when chlorophyllide as an amphiphilic substrate exists, so it is possible to selectively kill cells that express the multimeric protein. However, this patent publication merely mentions a function of the multimeric protein as a superoxide-generating reagent, but does not suggest details and schemes for practical application of such a protein to an antioxidant screening method.